The ability of cells to move and adhere to neighboring cells are two critical processes involved in embryonic development, wound healing, and tissue remodeling. Disruption of these two processes can have deleterious effects on cell behavior and lead to carcinogenesis, which is the basis of tumor formation and metastasis. Cell adhesion protein molecules (cadherins) and small GTPase proteins (Rac1) both play a critical role in initiating cell-cell adhesion. Cell migration involves constant remodeling of the actin cytoskeleton, which functions in many important cellular processes including cell movement and in the establishment and maintenance of cell- cell adhesions. Upon two cells coming together, the actin cytoskeleton becomes reorganized to allow for cell adhesion molecules (cadherins) to engage with similar molecules on an opposing contacting membrane. Cadherins mediate cell-cell adhesion by binding and clustering (i.e. recruiting) adjacent cadherin molecules. Rac1 also mediates cell-cell adhesion by switching between active (on) and inactive (off) conformations depending on signals from outside and inside the cell. During initial cell-cell adhesion, Rac1 localizes to expanding membrane protrusions and facilitates reorganization of the actin cytoskeleton. The long-term goal of this project is to understand how cadherins and Rac1 work together to mediate and strengthen initial cell-cell adhesions. Previous studies have shown that active Rac1 temporally localizes to sites of initial cell-cell contact where cadherin molecules are undergoing binding and clustering, but then becomes inactivated and removed as cell-cell adhesions are strengthened. The temporal localization of Rac1 to cell-cell adhesion sites containing clustered cadherins suggests that cadherins signal other molecules to regulate Rac1 activity levels. However, the proteins involved in activating or inactivating Rac1 during initial cell-cell adhesion remain undefined. In this proposal, the effect of cadherin binding and clustering on Rac1 activity levels during de novo cell-cell contact will be investigated using a newly developed experimental technique, specifically designed to examine cell-cell adhesion only. In addition, I aim to identify using proteomic and knockdown techniques the proteins involved in turning Rac1 on and off at initial cell-cell adhesion sites and the signaling intermediates that are activated upon cell-cell adhesion to regulate cadherin binding and recruitment of Rac1 regulator proteins. These findings will advance our understanding of the signaling pathways and protein complexes involved in normal cell function as well as in detecting cellular changes upon cell differentiation and carcinogenesis. PUBLIC HEALTH RELEVANCE: Any type of disruption of the cellular environment has the ability to not only change the adhesive property of the cell, but can contribute to aberrant cell migration and lead to tumorigenesis and/or metastasis. Recent studies demonstrate that overexpression of Rac1 leads to tumor formation, but the signals involved remain undetermined. The studies proposed in this project will advance our understanding of the molecular and cellular events involved in regulating Rac1 activity during cell-cell adhesion under normal conditions. In addition, it will shed new light on how overexpression or inhibition of specific proteins and/or signaling pathways can lead to aberrant protein localization and cancer initiation.